Vascular endothelial growth factor (VEGF) is a potent mitogen specific for endothelial cells. Its expression is dramatically induced by low oxygen tension in a variety of cell types, and it has been suggested to be a key mediator of hypoxia-induced angiogenesis. Although VEGF action is targeted to endothelial cells, it is generally believed that these cells do not express VEGF. In addition, the mechanisms by which hypoxia regulates VEGF production remain unclear. We report in the present study that pulmonary artery endothelial cells do not express VEGF under basal conditions; however, significant VEGF mRNA levels accumulate when these cells are exposed to hypoxia. Using a DNA fragment containing human VEGF promoter sequence, we identified a 28-bp element that is necessary and sufficient to upregulate transcription in response to hypoxia. This element can act as a hypoxia-specific enhancer when placed upstream or downstream from a heterologous promoter. The enhancer includes, in addition to an octamer homologous to the hypoxia-inducible factor-1 (HIF-1) consensus, a sequence that resides 3' to the consensus. Although this sequence may not be involved in the binding of HIF-1, it is absolutely required for the enhancer activity and may be the binding site for certain constitutive binding proteins. The expression of VEGF by endothelial cells in response to hypoxia may provide an important mechanism by which endothelial cell permeability and proliferation is regulated in an autocrine manner.

Background Hypoxia induces an inflammatory response in the lung manifested by alternative activation of macrophages with elevation of pro-inflammatory mediators that are critical for the later development of hypoxic pulmonary hypertension (HPH). Mesenchymal stromal cell (MSC) transplantation inhibits lung inflammation, vascular remodeling and right heart failure, and reverses HPH in experimental models of disease. In this study, we aimed to investigate the paracrine mechanisms by which MSCs are protective in HPH. Methods and Results We fractionated mouse MSC-conditioned media to identify the biologically-active component affecting in vivo hypoxic signaling and determined that exosomes, secreted membrane microvesicles, suppressed the hypoxic pulmonary influx of macrophages and the induction of pro-inflammatory and pro-proliferative mediators, including monocyte chemoattractant protein-1 and hypoxia-inducible mitogenic factor, in the murine model of HPH. Intravenous delivery of MSC-derived exosomes (MEX) inhibited vascular remodeling and HPH, whereas MEX-depleted media or fibroblast-derived exosomes had no effect. MEX suppressed the hypoxic activation of signal transducer and activator of transcription 3 (STAT3) and the upregulation of the miR-17 superfamily of microRNA clusters, whereas it increased lung levels of miR-204, a key microRNA whose expression is decreased in human PH. MEX produced by human umbilical cord MSCs inhibited STAT3 signaling in isolated human pulmonary artery endothelial cells demonstrating a direct effect of MEX on hypoxic vascular cells. Conclusions This study indicates that MEX exert a pleiotropic protective effect on the lung and inhibit PH through suppression of hyperproliferative pathways, including STAT-3 mediated signaling induced by hypoxia.